Experiments proposed here continue our investigation of the multiplex activities of signaling pathways that promote hypertrophy and alter the structure and function of cardiac myofilaments. Our hypothesis is that alterations in myofilament protein phosphorylation associated with hypertrophy and failure represent a significant factor limiting both contraction and relaxation reserve in humans. In new lines of experiments, we focus on novel signaling through effectors of the Rho subfamily of small G proteins including Rho dependent kinase (ROK) and p21-activated kinase (Pak 1). Our aims are: Aim #1 : To determine the steady state relation between Ca2+ and force/ATPase activity in myofilaments from normal (C) end-stage human failed hearts (HF), and supported HF (LVAD) before and after exchange of the troponin (cTn) complex with recombinant complex containing unphosphorylated or pseudo-phosphorylated cTnT and cTnl. Measurements are made with variations in pH and sarcomere length. Aim #2: To determine the mechanism for the altered cTn activity in C, H, and LVAD hearts by comparing phosphorylation of myofilament proteins (cTnl, cTnT, C-protein, myosin light chain 2). Aim #3: To determine acute effects of altered RhoA and ROK activity on rat and mouse cardiomyocyte function ((Ca2+)i and shortening), on myofilament tension cost and response to Ca2+, and on sites and relative levels of myofilament protein phosphorylation. Aim #4: To determine the mechanism by which activation of Pak 1 induces dephosphorylation of myofilament proteins and altered cellular function, and whether this mechanism is a significant mechanism in anti-adrenergic effects. We approach these aims using well-characterized samples of human myocardium, transgenic models, as well as adenoviral mediated transfer of cDNA to specifically activate the ROK and Pak 1 pathways. Methods include measurements of shortening and Ca2+ in intact myocytes and force, shortening, and ATPase rate in single myocytes and fiber bundles from control rats and mice and transgenic models deficient in functionally significant protein kinase A (PKA) and PKC myofilament sites, and phospholamban. Post-translational modifications are analyzed using phosphospecific antibodies, analytical electrophoresis, and mass spectrometry. Data from these experiments provide novel insights into the mechanisms of heart failure and potential therapies.